Evidence of Residual Ongoing Viral Replication in Chronic Hepatitis B Patients Successfully Treated With Nucleos(t)ide Analogues.

BACKGROUND: Chronic hepatitis B is usually treated with nucleos(t)ide analogues (NAs). However, a cure is rarely achieved, even with years of treatment. Here, we investigated whether viral replication is completely halted and how long covalently closed circular DNA (cccDNA) persists in patients successfully treated with NAs. METHODS: A series of longitudinal serum samples and a collection of cross-sectional liver biopsies were obtained from patients successfully treated with NAs. Viral variants in serum HBV RNA were enumerated by deep sequencing. Viral replication intermediates in hepatocytes were directly visualized by in situ hybridization. The apparent half-life of each cccDNA was estimated. RESULTS: Three of 6 successfully treated patients demonstrated clear evidence of a small proportion of virus evolution, although the overwhelming proportion of variants were identical or possessed a similar degree of divergence through time. The apparent half-life of variants was estimated to be from approximately 7.42 weeks to infinite. Hepatocytes remained positive for cytoplasmic nucleocapsids-associated relaxed circular DNA in 4 of 7 liver needle biopsies. CONCLUSIONS: We conclude that even after prolonged treatment, a small proportion of the cccDNA reservoir is constantly replenished by continued low-level HBV replication, whereas a large proportion of the cccDNA reservoir persists over time.

Roxadustat promotes hypoxia-inducible factor-1α/vascular endothelial growth factor signalling to enhance random skin flap survival in rats.

Random skin flaps have limited clinical application as a broad surgical reconstruction treatment because of distal necrosis. The prolyl hydroxylase domain-containing protein inhibitor roxadustat (RXD) enhances angiogenesis and reduces oxidative stress and inflammation. This study explored the function of RXD in the survival of random skin flaps. Thirty-six male Sprague-Dawley rats were randomly divided into low-dose RXD group (L-RXD group, 10 mg/kg/2 day), high-dose RXD group (H-RXD group, 25 mg/kg/2 day), and control group (1 mL of solvent, 1:9 DMSO:corn oil). The proportion of surviving flaps was determined on day 7 after surgery. Angiogenesis was assessed by lead oxide/gelatin angiography, and microcirculation blood perfusion was evaluated by laser Doppler flow imaging. Specimens in zone II were obtained, and the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured as indicators of oxidative stress. Histopathological status was evaluated with haematoxylin and eosin staining. The levels of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and the inflammatory factors interleukin (IL)-1ß, IL-6, and tumour necrosis factor-α (TNF-α) were detected by immunohistochemistry. RXD promoted flap survival and microcirculatory blood perfusion. Angiogenesis was detected distinctly in the experimental group. SOD activity increased and the MDA level decreased in the experimental group. Immunohistochemistry indicated that the expression levels of HIF-1α and VEGF were increased while the levels of IL-6, IL-1ß, and TNF-α were decreased after RXD injection. RXD promoted random flap survival by reinforcing vascular hyperplasia and decreasing inflammation and ischaemia-reperfusion injury.

A link between IL-23 and anti-CD4 autoantibody production in antiretroviral-treated HIV-infected individuals.

Potential mechanisms of poor CD4+ T cell reconstitution after viral suppression with antiretroviral therapy (ART) in HIV disease have been extensively investigated. We recently discovered that anti-CD4 autoantibody plays a role in impaired CD4+ T cell recovery from ART in HIV-infected individuals with viral suppression, which accounts for a mechanism specific for CD4+ T cell depletion. However, the mechanism of pathologic anti-CD4 autoantibody production in treated HIV disease remains unknown. Here we report that seasonal influenza vaccination induced IgG anti-CD4 autoantibodies, predominant IgG3 subclass, in some viral-suppressed ART-treated HIV+ subjects. To explore the mechanism of anti-CD4 antibody production in this population, we performed and analyzed gene profiles in isolated B cells using a gene microarray and plasma 32 cytokines. Notably, both gene expression and multiple cytokine analyses showed pre-vaccination plasma level of IL-23 was the key cytokine linked to IgG anti-CD4 antibody production in response to immunization in vivo Exogenous rIL-23 increased autoreactive IgG binding on CD4+ T cells from HIV+ subjects in vitro Results from this study may reveal a role of IL-23 in anti-CD4 autoantibody production in treated HIV.IMPORTANCEIn our published studies, we determine that pathological anti-CD4 IgGs from immunologic non-responders on virally-suppressive ART (CD4 cell counts < 350 cells/µL) mediated CD4+ T cell death via antibody-mediated cytotoxicity (ADCC), which play a role in poor CD4+ T cell recovery from ART. Up to 25% of HIV-infected individuals are non-responders and demonstrate increased morbidity and mortality. However, the mechanism of anti-CD4 autoantibody production in treated HIV remains unknown. In this study, we report that IL-23 may be the key cytokine to promote anti-CD4 autoantibody production after immunization in ART-treated HIV-infected individuals.

TIPE1 impairs stemness maintenance in colorectal cancer through directly targeting ß-catenin.

TIPE1 (tumor necrosis factor-α-induced protein 8-like 1) contributes to cell death in diverse cancers. However, the expression and biological functions of TIPE1 in colon cancer remain unclear. In the present study, we report that TIPE1 was downregulated in colon cancer tissues and positively correlates with prognosis of colon cancer patients. TIPE1 overexpression significantly inhibits colon cancer cell growth both in vitro and in vivo through impairing stemness, accompanied with downregulation of the stemness-related markers, ALDH, CD133, CD44 and SOX-9. Mechanically, TIPE1 directly targets ß-catenin and promotes ß-catenin degradation in a protease-dependent manner, and Wnt/ß-catenin signaling plays a crucial role during TIPE1-mediated stemness inhibition in colon cancer. These findings reveal that TIPE1 exerts anti-tumor effects in colon cancer and suggest that TIPE1 would be a therapeutic target for cancers.

Relationship between serum HBV-RNA levels and intrahepatic viral as well as histologic activity markers in entecavir-treated patients.

BACKGROUND & AIMS: In diagnostics, serum hepatitis B virus (HBV)-RNA levels are valuable when the HBV-DNA load in circulation is effectively suppressed by nucleos(t)ide analogue (NUC) therapy. This study aimed to determine the intrahepatic viral replication activity reflected in serum HBV-RNA and whether HBV-RNA contributes to liver histological changes in patients treated with NUC. METHODS: A cross-sectional set of serum and liver biopsy samples was obtained from patients treated with entecavir, who had undetectable levels of serum HBV-DNA. The correlations between serum HBV-RNA concentration and levels of peripheral and intrahepatic viral replicative forms, as well as histological scores, were analyzed. Quasispecies of serum HBV-RNA and intrahepatic viral replicative forms were examined by deep sequencing. HBV-RNA-positive hepatocytes were visualized by in situ hybridization. RESULTS: Serum HBV-RNA was detected in 35 of 47 patients (74.47%, 2.33-4.80log10copies/ml). These levels correlated not only with the intrahepatic HBV-RNA level and the ratio of intrahepatic HBV-RNA to covalently closed circular DNA (cccDNA), but also with the histological scores for grading and staging. Regarding quasispecies, serum HBV-RNA was dynamic and more genetically homogenous to simultaneously sampled intrahepatic HBV-RNA than to the cccDNA pool. In situ histology revealed that HBV-RNA-positive hepatocytes were clustered in foci, sporadically distributed across the lobules, and co-localized with hepatitis B surface antigen. CONCLUSION: Serum HBV-RNA levels reflect intrahepatic viral transcriptional activity and are associated with liver histopathology in patients receiving NUC therapy. Our study sheds light on the nature of HBV-RNA in the pathogenesis of chronic HBV infection and has implications for the management of chronic hepatitis B during NUC therapy. LAY SUMMARY: Serum HBV-RNA levels are indicative of the intrahepatic transcriptional activity of covalently closed circular DNA and are associated with liver histological changes in patients with chronic B hepatitis who are receiving nucleos(t)ide analogue therapy.

Nesfatin-1 regulates the HMGB1-TLR4-NF-κB signaling pathway to inhibit inflammation and its effects on the random skin flap survival in rats.

OBJECTIVE: Random skin flaps are often placed by plastic surgeons to treat limb deformities and dysfunction. Nesfatin-1 (NES) is a peptide that exerts angiogenic, anti-inflammatory, and anti-oxidant effects. We assessed the impact of NES on flap survival and the underlying mechanism. METHODS: We modified the McFarlane random skin flap rat model. Thirty-six male Sprague-Dawley rats were randomly divided into a control group (corn oil solution with DMSO), low-dose group (NES-L at 10 µg/kg/day), and high-dose group (NES-H at 20 µg/kg/day). On day 7 after surgery, average flap survival areas were calculated. Laser Doppler blood flow monitoring and lead oxide/gelatin angiography were used to evaluate blood perfusion and neovascularization, respectively. Flap histopathological status was evaluated by hematoxylin and eosin (H&E) staining. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were determined. Immunohistochemical techniques were used to evaluate the expression of angiogenetic and inflammatory factors. RESULTS: In the experimental groups, the mean skin flap survival areas and blood perfusion increased considerably. The SOD activities in the experimental groups increased and the MDA contents decreased. Immunohistochemically, VEGF expression was upregulated in the experimental groups and the expression levels of inflammatory factors decreased markedly. CONCLUSION: NES inhibited ischemic skin flap necrosis, promoted angiogenesis, and reduced ischemia-reperfusion injury and inflammation. Inhibition of the inflammatory HMGB1-TLR4-NF-κB signal pathway, which reduced flap inflammation and oxidative stress, may explain the enhanced flap survival.

Saxagliptin promotes random skin flap survival.

BACKGROUND: Flap necrosis is a common issue encountered in clinical flap transplantation surgery. Here, we assessed the effects of saxagliptin, a dipeptidyl peptidase-4 inhibitor, on flap survival and explored the underlying mechanisms. METHODS: A dorsal McFarlane flap model was established in 36 rats, which were randomly divided into a high-dose saxagliptin (HS) group (saxagliptin, 30 mg/kg/day, n = 12), low-dose saxagliptin (LS) group (saxagliptin, 10 mg/kg/day, n = 12), and control group (n = 12). On day 7, flap survival was examined by eye in six rats from each group, along with determination of blood perfusion by laser Doppler flowmetry and angiogenesis by angiography. The remaining rats were sacrificed for harvesting of flap tissue. The status of the flap tissue was examined histopathologically by staining with hematoxylin and eosin (H&E). Oxidative stress was evaluated by determination of superoxide dismutase (SOD) activity and malonaldehyde (MDA) content. Gasdermin D (GSDMD), vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), NOD-like receptor pyrin domain containing 3 (NLRP3), interleukin (IL)-6, IL-18, Toll-like receptor 4 (TLR4), IL-1ß, caspase-1, and nuclear factor-κB (NF-κB) expression were detected by immunohistochemical analysis. RESULTS: The experimental group exhibited a larger area of flap survival, with more blood perfusion and neovascularization and better histopathological status than the control group. The degree of oxidative stress and the levels of NF-κB, TLR4, proinflammatory cytokines, and pyroptosis-associated protein were decreased in the experimental group, while the VEGF level was increased in a saxagliptin dose-dependent manner. CONCLUSION: Saxagliptin promotes random skin flap survival.

Matrine suppresses NLRP3 inflammasome activation via regulating PTPN2/JNK/SREBP2 pathway in sepsis.

BACKGROUND: Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Abnormal activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome plays a vital role in the pathogenesis of sepsis. Matrine is proved to show good anti-inflammatory properties, whereas its effect and the underlying molecular machinery on sepsis remains unclear. PURPOSE: The aim of this study is to evaluate the effect and mechanism of Matrine on sepsis. STUDY DESIGN: THP-1 cells and J774A.1 cells were stimulated by lipopolysaccharide (LPS) with nigericin or adenosine triphosphate (ATP) to establish an in vitro model. Cecal ligation and puncture (CLP)-induced sepsis mouse model was used. Matrine was given by gavage. METHODS: To investigate the NLRP3 inflammasome activation, phorbol myristate acetate (PMA)-induced THP-1 cells were first primed with LPS and then stimulated by matrine, followed by treatment with nigericin or ATP. The concentration of interleukin 1ß (IL-1ß) and interleukin 18 (IL-18) in the cell culture supernatant was detected. The mechanism was explored by cell death assay, immunoblots and immunofluorescence in vitro. C57BL/6 mice were intragastrically administered with matrine for 5 days before CLP. The therapeutic effect of matrine was evaluated by symptoms, pathological analysis, ELISA and RT-qPCR. RESULTS: Our results revealed that matrine inhibited IL-1ß and IL-18 secretion, suppressed caspase-1 activation, reduced cell death, and blocked ASC speck formation upon NLRP3 inflammasome activation. Furthermore, matrine restrains NLRP3 inflammasome activation as well as pyroptosis through regulating the protein tyrosine phosphatase non-receptor type 2 (PTPN2)/JNK/SREBP2 signaling. Matrine also prominently improved the symptoms and pathological changes with reduced levels of TNF-α, IL-1ß, and IL-6 in the lung tissues and serum in a dose-dependent manner. CONCLUSION: Matrine effectively alleviates the symptoms of CLP-induced sepsis in mice, restrains NLRP3 inflammasome activation by regulating PTPN2/JNK/SREBP2 signaling pathway, and may become a promising therapeutic agent for sepsis treatment.

Activation of aldehyde dehydrogenase-2 improves ischemic random skin flap survival in rats.

Objective: Random skin flaps have many applications in plastic and reconstructive surgeries. However, distal flap necrosis restricts wider clinical utility. Mitophagy, a vital form of autophagy for damaged mitochondria, is excessively activated in flap ischemia/reperfusion (I/R) injury, thus inducing cell death. Aldehyde dehydrogenase-2 (ALDH2), an allosteric tetrameric enzyme, plays an important role in regulating mitophagy. We explored whether ALDH2 activated by N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide (Alda-1) could reduce the risk of ischemic random skin flap necrosis, and the possible mechanism of action. Methods: Modified McFarlane flap models were established in 36 male Sprague-Dawley rats assigned randomly to three groups: a low-dose Alda-1 group (10 mg/kg/day), a high-dose Alda-1 group (20 mg/kg/day) and a control group. The percentage surviving skin flap area, neutrophil density and microvessel density (MVD) were evaluated on day 7. Oxidative stress was quantitated by measuring the superoxide dismutase (SOD) and malondialdehyde (MDA) levels. Blood perfusion and skin flap angiogenesis were assessed via laser Doppler flow imaging and lead oxide-gelatin angiography, respectively. The expression levels of inflammatory cytokines (IL-1ß, IL-6, and TNF-α), vascular endothelial growth factor (VEGF), ALDH2, PTEN-induced kinase 1 (PINK1), and E3 ubiquitin ligase (Parkin) were immunohistochemically detected. Indicators of mitophagy such as Beclin-1, p62, and microtubule-associated protein light chain 3 (LC3) were evaluated by immunofluorescence. Results: Alda-1 significantly enhanced the survival area of random skin flaps. The SOD activity increased and the MDA level decreased, suggesting that Alda-1 reduced oxidative stress. ALDH2 was upregulated, and mitophagy-related proteins (PINK1, Parkin, Beclin-1, p62, and LC3) were downregulated, indicating that ALDH2 inhibited mitophagy through the PINK1/Parkin signaling pathway. Treatment with Alda-1 reduced neutrophil infiltration and expressions of inflammatory cytokines. Alda-1 significantly upregulated VEGF expression, increased the MVD, promoted angiogenesis, and enhanced blood perfusion. Conclusion: ALDH2 activation can effectively enhance random skin flap viability via inhibiting PINK1/Parkin-dependent mitophagy. Moreover, enhancement of ALDH2 activity also exerts anti-inflammatory and angiogenic properties.

A SIRPαFc Fusion Protein Conjugated With the Collagen-Binding Domain for Targeted Immunotherapy of Non-Small Cell Lung Cancer.

The SIRPαFc fusion protein can block the immunosuppressive CD47-SIRPα signal between macrophages and tumor cells as a decoy receptor and has demonstrated its immunotherapeutic efficacy in various tumors. However, its clinical application was limited because of the potential hematologic toxicity. The heptapeptide "TKKTLRT" is a collagen-binding domain (CBD) which can bind collagen specifically. Herein, we aim to improve the tumor targeting of SIRPαFc and therefore avoid its unnecessary exposure to normal cells through synthesizing a TKKTLRT-SIRPαFc conjugate. Experiments at molecular and cellular levels indicate that the TKKTLRT-SIRPαFc conjugate-derived collagen-binding affinity and the introduction of CBD did not impact the CD47-binding affinity as well as its phagocytosis-promoting effect on NSCLC cells. In vivo distribution experiments showed that CBD-SIRPαFc accumulated in tumor tissue more effectively compared to unmodified SIRPαFc, probably due to the exposed collagen in the tumor vascular endothelium and stroma resulting from the abnormal vessel structure. On an A549 NSCLC nude mouse xenograft model, CBD-SIRPαFc presented more stable and effective antitumor efficacy than SIRPαFc, along with significantly increased CD11b+F4/80+ macrophages especially MHC II+ M1 macrophages within tumors. All of these results revealed that CBD brought a tumor-targeting ability to the SIRPαFc fusion protein, which contributed to the enhanced antitumor immune response. Altogether, the CBD-SIRPαFc conjugate may have the potential to be an effective tumor immunotherapy with improved antitumor efficacy but less non-tumor-targeted side effect.

Transcriptional analysis of immune-related genes in dendritic cells from hepatitis B surface antigen (HBsAg)-positive transgenic mice and regulation of Fc gamma receptor IIB by HBsAg-anti-HBs complex.

To study the gene expression profiles in dendritic cells (DCs) from a hepatitis B surface antigen (HBsAg) positive host, transcriptional analysis of bone marrow -derived DCs from a lineage of HBsAg transgenic mice (#59) was compared to DCs from normal mice. Among the immune-related genes, 12 were up-regulated, and 14 were down-regulated in transgenic mice relative to those of normal mice. The up-regulated genes include genes encoding immunoglobulin, histocompatibility 2 (K region), and several complement component genes, while the down-regulated genes include the TAP1 (transporters associated with antigen processing gene-1), interferon induced gene (Ifi203), chemokine (C-X-C) ligands and leukocyte-immunoglobulin-like genes, Lck-interacting transmembrane adaptor genes and histocompatibility 2 (Q region and T region). Since an immunogenic complex containing HBsAg-anti-HBs has been used as a therapeutic vaccine for clinical trial in chronic hepatitis B patients, DCs from #59 were incubated with immunogenic complex compared to those incubated with HBsAg alone. The immune-related six genes up-regulated with immunogenic complex treatment were Fcgr2b, Cxcl2, Fth1, Clec4n, Lilrb4, and Dbh, with Fcgr2b (Fc gamma receptor IIB) being the highest up-regulated gene. Interestingly, levels of Fcgr2b were found up-regulated in patients with chronic hepatitis B undergoing immunogenic complex immunization, which returned to baseline when immunization was discontinued. In conclusion, by transcriptional analysis, immunogenic complex induced up-regulation of Fcgr2b expression both in dendritic cells from an HBsAg transgenic mouse model and peripheral B cells from patients with chronic hepatitis B, which indicates that Fcgr2b is one of the key molecules up-regulated by immunogenic complex.

Frequently Transmission and Close Relationship Among Immigrants in the China-Myanmar Border Region Indicated by Molecular Transmission Analysis From a Cross-Sectional Data.

Background: Accurate identification of molecular transmission clusters (MTCs) and understanding the dynamics of human immunodeficiency virus (HIV) transmission are necessary to develop targeted interventions to prevent HIV transmission. We evaluated the characteristics of antiretroviral therapy-naïve individuals who belonged to HIV-1 MTCs in the China-Myanmar border region to inform targeted effective HIV intervention. Methods: Phylogenetic analyses were undertaken on HIV-1 pol sequences to characterize subtypes or circulating recombinant forms and identify MTCs. MTCs were defined as those with 2 or more sequences having bootstrap support > 80% and a pairwise gene distance less than or equal to 0.03. Factors correlated with MTCs were evaluated using logistic regression analysis. The chi-square test was used to compare differences between Chinese and Burmese participants belonging to MTCs. Results: A total of 900 people had their pol gene successfully sequenced. Twenty-one MTCs were identified and included 110 individuals (12.2%). Individuals in MTCs were more likely to be Burmese [aOR = 2.24 (95% CI: 1.33, 3.79), P = 0.003], be younger [aOR = 0.34 (95% CI: 0.20, 0.58), P < 0.001 for age 26-50 vs. 25 years or younger], have a lower CD4 T cell count [aOR = 2.86 (95% CI: 1.34, 6.11), P = 0.007 for < 200 vs. 350 or greater], and have subtypes CRF07_BC or C [CRF07_BC: aOR = 7.88 (95% CI: 3.55, 17.52), P < 0.001; C: aOR = 2.38 (95% CI: 1.23, 4.62), P = 0.010 compared to CRF01_AE]. In MTCs, Burmese were younger (89.7 vs. 57.7% for age 25 years or younger), had a lower education level (41.0 vs. 8.5% for illiterate), were more likely to be infected through injection drug use (35.9 vs. 12.7%), and had a higher proportion of subtype BC (33.3 vs. 15.5%) and CRF01_AE (20.5 vs. 8.5%) compared to Chinese (P < 0.05 for all). Conclusion: Burmese participants were more likely to belong to MTCs, and most MTCs had both Burmese and Chinese participants. These data highlight the bidirectional transmission of HIV-1 frequently transmission and close relationship among immigrants in the China-Myanmar border region. Local health departments should pay more attention to HIV screening and intervention to immigrants Burmese with the characteristics of younger age, having lower CD4 T cell count and infected with HIV subtypes CRF07_ BC or C.

Analysis of Vpr Genetic Variations between Chinese Major Circulating Recombinants CRF01_AE and CRF07_BC.

BACKGROUND: HIV-1 CRF01_AE and CRF07_BC recombinant strains are responsible for more than 80% of new infections in China since the beginning of the 2000s. These two strains may have distinct genetic mutations, which resulted in distinct patterns of pathogenesis related to the viral gene, Vpr. OBJECTIVE: The amino acid pattern and genetic diversity of Vpr were analyzed and characterized in HIV-1 CRF01_AE and CRF07_BC HIV-1 strains. METHODS: The Vpr gene was amplified from extracted viral RNA and DNA sequencing was performed using an ABI3730 analyzer. The positional amino acid composition, genetic variation and distance of Vpr sequence were analyzed by Bio-Edit 7.2 and Mega 6.01 software packages. RESULTS: A total of 162 CRF01_AE and 80 CRF07_BC derived Vpr sequences were obtained by DNA sequencing. CRF01_AE patients showed higher viral load and lower CD4 counts than CRF07_BC patients (P<0.05). Higher genetic distance and more polymorphic amino acids were found in CRF01_AE Vpr than CRF07_BC Vpr (P<0.05). The common conservative amino acid region was identified as 29EAVRHFP35 in both CRF07_BC and CRF01_AE. Of note, the R77Q mutation was found in both the most recently Chinese derived CRF07_BC and CRF01_AE Vpr. CONCLUSION: CRF01_AE derived Vpr has higher genetic variation and pathogenesis in comparison to the CRF07_BC strain.

Glucocorticoids Regulate the Activation of Neutrophils and Inhibit the Formation of Pulmonary Embolism.

BACKGROUND: There is a close relationship between neutrophil extracellular traps (NETs) and venous thromboembolism (VTE). The regulatory role and mechanism of glucocorticoids (GC) in the formation of NETs are unclear. OBJECTIVE: This study was conducted to assess the effect of GC on the formation of NETs. METHODS: We constructed a mouse VTE model and treated them with GC to observe the effect of GC on the formation of NETs. In this regard, peripheral blood neutrophils were isolated, and the effect and mechanism of GC in neutrophil activation were analyzed. RESULTS: Following LPS treatment, the colony-forming ability of neutrophils and their ability to form NETs increased significantly. The analysis of cytokine changes by RT-PCR combined with ELISA showed that the level of inflammatory factors in LPS-activated neutrophils increased significantly; however, these factors were significantly inhibited after GC treatment, and the inhibitory effect was positively correlated with the concentration of GC. LPS treatment was able to activate the production of ROS and lipid peroxides, however, this activation was significantly inhibited after GC treatment, and the inhibition increased with increasing doses of GC. Further examination of the changes in NF-κB signaling activation revealed that LPS-induced NF-κB signaling was significantly inhibited after GC treatment, and this inhibition increased with increasing the GC concentration. CONCLUSION: Glucocorticoids were able to inhibit neutrophil activation and reduce the formation of NETs. The research results provided a new research direction for clinical antithrombotic treatment.

Phenotypic and genotypic characterization of human immunodeficiency virus type 1 CRF07_BC strains circulating in the Xinjiang Province of China.

BACKGROUND: HIV-1 CRF07_BC recombinant previously circulated mainly among the intravenous drug users (IDUs) in Xinjiang province of China and is currently spreading in the entire country. The aim of this study is to characterize the genotypic and phenotypic properties of HIV-1 CRF07_BC isolates in comparison with those of the subtype B' (Thailand B) which is prevalent in the former plasma donors (FPDs) in China. RESULTS: Twelve HIV-1 CRF07_BC variants were isolated from the blood of the HIV-1-infected IDUs in Xinjiang province, and 20 subtype B' isolates were obtained from the FPDs in Anhui and Shanxi provinces of China. All the CRF07_BC viruses utilized CCR5 co-receptor, whereas 12 subtype B' viruses were R5-tropic, and the remaining B' isolates were dual (R5X4) tropic. CRF07_BC viruses had lower net charge value in the V3 loop and exhibited slower replication kinetics than subtype B' viruses. The number and location of the potential N-linked glycosylation sites in V1/V2 and the C2 region of the CRF07_BC viruses were significantly different from those of the subtype B' viruses. CONCLUSION: The HIV-1 CRF07_BC recombinant strains with relatively lower net charges in the V3 loop exclusively utilize CCR5 co-receptor for infection and exhibit slow replication kinetics in the primary target cells, suggesting that CRF07_BC may be superior over B' and other HIV-1 subtypes in initiating infection in high-risk population. These findings have molecular implications for the adaptive evolution of HIV-1 circulating in China and the design of tailored therapeutic strategy for treatment of HIV-1 CRF07_BC infection.

[Primers of gag gene for HIV-1 subtyping in China and application thereof in practice].

OBJECTIVE: To design the appropriate primers of gag gene for HIV-1 subtyping in molecular epidemiology survey based on the genetic characteristics derived from the main HIV-1 strains prevailing in China. METHODS: The gag genes of HIV-1 CRF07_BC, CRF08_BC, and subtype C, together with subtyping reference sequences, were obtained from HIV sequence database. Referring to the alignments and genetic characteristics of HIV-1 full gag sequences, new primers of gag gene for amplification and subtyping were designed. The target fragment was used to construct neighbor-joining phylogenetic tree and evaluate its reliability. The newly designed primers (GUX/GDX) were used to amplify the plasma samples to evaluate their efficiency. RESULTS: The phylogenetic tree of 306/c-gag fragments (positions 836-1507 of HIV-1 strain HXB2) showed that CRF07_BC and subtype C strains formed clusters with low bootstrap values (59% for CRF07_BC and 70% for subtype C), and the phylogenetic tree could not distinguish the sequences of CRF07_BC, CRF08_BC, and subtype C very well. Whereas the sequences of CRF07_BC, CRF08_BC, and subtype C from GUX/GDX (positions 781-1861) were clustered separately with higher bootstrap values (99%, 99%, and 77% respectively). In practice, a very good amplification and sequencing efficiency with over 90% positive results on average were obtained with GUX/GDX. Five clusters of subtype B, C, CRF01_AE, CRF07_BC, and CRF08_BC were formed with higher confidence (Bootstrap values all above 80%). The reliable phylogenetic tree could be constructed based on the fragments sequenced only with antisense primer (GDX). CONCLUSION: Fragments obtained with GUX/GDX primers of gag gene can be used to reconstruct phylogenetic tree with high reliability to distinguish the HIV-1 strains circulating in China, especially for the major BC recombinant and subtype C strains, which provides a useful tool in HIV molecular epidemiologic research.

A robust 6-mRNA signature for prognosis prediction of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal malignancies worldwide. PDAC prognostic and diagnostic biomarkers are still being explored. The aim of this study is to establish a robust molecular signature that can improve the ability to predict PDAC prognosis. 155 overlapping differentially expressed genes between tumor and non-tumor tissues from three Gene Expression Omnibus (GEO) datasets were explored. A least absolute shrinkage and selection operator method (LASSO) Cox regression model was employed for selecting prognostic genes. We developed a 6-mRNA signature that can distinguish high PDAC risk patients from low risk patients with significant differences in overall survival (OS). We further validated this signature prognostic value in three independent cohorts (GEO batch, P < 0.0001; ICGC, P = 0.0036; Fudan, P = 0.029). Furthermore, we found that our signature remained significant in patients with different histologic grade, TNM stage, locations of tumor entity, age and gender. Multivariate cox regression analysis showed that 6-mRNA signature can be an independent prognostic marker in each of the cohorts. Receiver operating characteristic curve (ROC) analysis also showed that our signature possessed a better predictive role of PDAC prognosis. Moreover, the gene set enrichment analysis (GSEA) analysis showed that several tumorigenesis and metastasis related pathways were indeed associated with higher scores of risk. In conclusion, identifying the 6-mRNA signature could provide a valuable classification method to evaluate clinical prognosis and facilitate personalized treatment for PDAC patients. New therapeutic targets may be developed upon the functional analysis of the classifier genes and their related pathways.

Systemic translocation of Staphylococcus drives autoantibody production in HIV disease.

BACKGROUND: Increased autoreactive antibodies have been reported in HIV disease; however, the mechanism accounting for autoantibody induction in HIV remains unknown. RESULTS: Herein, we show that seasonal influenza vaccination induces autoantibody production (e.g., IgG anti-nuclear antibody (ANA) and anti-double-stranded DNA antibody (anti-dsDNA)) in some viral-suppressed antiretroviral therapy (ART)-treated HIV+ subjects, but not in healthy controls. These autoantibodies were not derived from antigen-specific B cells but from activated "bystander" B cells analyzed by single-cell assay and by study of purified polyclonal ANAs from plasma. To explore the mechanism of autoantibody generation in HIV+ subjects, plasma level of microbial products, gene expression profile of B cells, and B cell receptor (BCR) repertoires were analyzed. We found that autoantibody production was associated with increased plasma level of microbial translocation; the patients with high autoantibodies had skewed B cell repertoires and upregulation of genes related to innate immune activation in response to microbial translocation. By analyzing circulating microbial 16S rDNA in plasma, the relative abundance of Staphylococcus was found to be associated with autoantibody production in HIV+ subjects. Finally, we found that injection of heat-killed Staphylococcus aureus promoted germinal center B cell responses and autoantibody production in mice, consistent with the notion that autoantibody production in HIV+ patients is triggered by microbial products. CONCLUSIONS: Our results showed that translocation of Staphylococcus can promote B cell activation through enhancing germinal center response and induces autoantibody production. It uncovers a potential mechanism linking microbial translocation and autoimmunity in HIV+ disease and provides a strong rationale for targeting Staphylococcus to prevent autoantibody production.

Construction and characterization of an infectious molecular clone of HIV type 1 CRF07_BC.

HIV-1 CRF07_BC constitutes a large fraction of HIV-1 strains circulating in China and is responsible for the rapidly expanding epidemic across the country. Little is known about the biological characteristics of the CRF07_BC viruses, particularly the propensity by which this circulating recombinant form of HIV replicates within an infected individual. In this study, a near-full-length genome of a CRF07_BC virus from an injecting drug user (IDU) from the Xingjiang region in China was cloned and rescued by the functional 5' LTR (long terminal repeat region) and 3' LTR from pNL4-3 to produce a chimeric infectious molecular clone (NLXJDC6441X2). When cultured in interleukin-2-stimulated peripheral blood mononuclear cells (PBMCs) and the Ghost.R5 cell line, NLXJDC6441X2 produced a lower replication yield than expected. More study is needed to explore the key role that leads to the poor infectious activity of NLXJDC6441X2. This study has produced the first isolation of the HIV-1 CRF07_BC DNA clone and has provided a versatile molecular model for research focusing on the biological properties of this subtype.